The liberation of calcium ions from intracellular stores into the cytosol is used as a signaling mechanism by virtually all cell types to regulate functions as diverse as secretion, contraction, proliferation, and cell death. Advances in observational techniques have revealed complex patterns of intracellular Ca2+ that serve to selectively regulate specific cellular responses, and are constructed hierarchically through the activity of individual ion channels, multiple channels within clusters, and interactions between clusters. It is impossible to resolve all these scales simultaneously in a single experiment and the shorter time and distance scales cannot be resolved by any available experimental approaches. We therefore propose to use a tightly integrated approach of modeling, electrophysiology, and high-resolution cellular calcium imaging to illuminate the mechanisms by which "elementary" Ca2+ events are triggered and coupled to produce global cellular calcium signals. Specific aims are to describe (i) the kinetic mechanisms underlying the flux of calcium through single channels, (ii) the functional coupling between individual channels within a cluster, and (iii) the coordination between clusters allowing the propagation of global signals and the effects of buffers on this process. Numerical models will be constructed based on hypotheses and parameter values derived from patch-clamp and confocal cellular imaging experiments, and will be iteratively tested by comparison with observations and by their predictive power. Our overall goal is to develop a comprehensive model of intracellular Ca2+ signaling that is consistent with a multitude of observations, has predictive value, and extends to crucial - but experimentally inaccessible -space and time scales (nanometers and microseconds). We will focus on inositol trisphosphate-mediated Ca2+ signaling, utilizing Xenopus oocytes as a well-characterized model system, but the emergent principles will be widely applicable across many cell types and species, as well as to calcium signalling mediated by ryanodine receptors.